Early Dark Period Research Articles

The active phase-related increase in corticosterone and aggression are linked.

Recently we demonstrated that corticosterone exerts an acute facilitatory effect on aggression in male rats. Corticosterone production reaches a maximum at the onset of the dark period, while male rats are more aggressive in the dark. Here we present evidence demonstrating that the corticosterone increase at the beginning of the dark period is causally linked to the increase in aggressiveness. We measured plasma corticosterone and quantified aggressive behaviour of male territorial rats at various time points of the day-night transition. Low aggression levels were observed in the full light period when plasma concentrations of corticosterone were low. An increase in plasma corticosterone occurred just prior to the dark phase, when aggressive responding was the highest. Aggressive behaviour remained high in the early dark period when corticosterone was still high. We found that blocking the high affinity mineralocorticoid receptor (MR) with spironolactone (5 or 10 mg/kg) during the early dark period dramatically and specifically reduced territorial aggression.

Transcriptional and translational regulation of nitrogenase in light-dark- and continuous-light-grown cultures of the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142.

Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium which demonstrated extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N2-fixing conditions. N2 fixation and respiration peaked at 24-h intervals early in the dark or subjective-dark period, whereas photosynthesis was approximately 12 h out of phase and peaked toward the end of the light or subjective-light phase. Gene regulation studies demonstrated that nitrogenase is carefully controlled at the transcriptional and posttranslational levels. Indeed, Cyanothece sp. strain ATCC 51142 has developed an expensive mode of regulation, such that nitrogenase was synthesized and degraded each day. These patterns were seen when cells were grown under either light-dark or continuous-light conditions. Nitrogenase mRNA was synthesized from the nifHDK operon during the first 4 h of the dark period under light-dark conditions or during the first 6 h of the subjective-dark period when grown in continuous light. The nitrogenase NifH and NifDK subunits reached a maximum level at 4 to 10 h in the dark or subjective-dark periods and were shown by Western blotting and electron microscopy immunocytochemistry to be thoroughly degraded toward the end of the dark periods. An exception is the NifDK protein (MoFe-protein), which appeared not to be completely degraded under continuous-light conditions. We hypothesize that cellular O2 levels were kept low by decreasing photosynthesis and by increasing respiration in the early dark or subjective-dark periods to permit nitrogenase activity. The subsequent increase in O2 levels resulted in nitrogenase damage and eventual degradation.

Development of Vasoactive Intestinal Peptide mRNA Rhythm in the Rat Suprachiasmatic Nucleus

Development of the daily rhythm of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN), a main locus of circadian oscillation, was investigated by in situ hybridization. The phenotypic expression of VIP neurons occurred in two developmental stages in the ventrolateral portion of the SCN (VLSCN): the first was found before birth in the rostral part, and the second occurred in the main part between postnatal day (P) 10 and P20. The latter period coincided with the time that the massive VIP-efferent fibers project to the subparaventricular zone. In the adult and P20, the VIP mRNA signals of the SCN showed a clear diurnal rhythm with a trough in the light phase and a peak in the dark phase under light/dark (LD) conditions, but under constant dark (DD) conditions, no VIP mRNA fluctuations were observed. At P10, however, it was found that SCN VIP mRNA showed a peak at the transition from night to day and a trough at early dark period in LD conditions, in sharp contrast to the night peak in the adult rhythm. In DD conditions, a light-phase peak and a dark-phase trough were also observed at P10, contrasting the arrhythmic feature at adult stage. The present findings suggest that daily VIP rhythm was first generated in the early developed clock-controlled rostral SCN neurons, and later regulated by light-dependent main VLSCN neurons.

Baroreceptor regulation of salt intake.

Baroreceptor input plays a critical role in body fluid balance and the endocrine response to NaCl consumption [25]. Experiments were performed to characterize the alterations in salt intake that are seen after baroreceptor denervation. Using chronically baroreceptor denervated (SAD) or control (CON) male Sprague-Dawley rats, we determined: 1) concentration-dependent consumption of NaCl, 2) time course of saline intake, 3) effect of food access on saline intake, 4) intake of sucrose vs. saline, and 5) water vs. saline intake using a choice paradigm. In protocols 1-4 the rats were given a single bottle containing saline or sucrose for a 2-h period during the early dark period. A comparison of the intake of varying concentrations of NaCl (0.3 to 2.0% NaCl, six concentrations) demonstrated that the SAD consumed significantly less NaCl than the CON (from 0.9 to 2% NaCl), Saline Intake in SAD was 14-56% of the CON (significant group, salt concentration and interaction effects). Regression analysis demonstrated that in the SAD there was an inverse relationship between concentration and the amount of NaCl consumed (p < 0.02), an effect not seen in the CON. There were also differences in the pattern of saline intake with the CON showing the highest consumption in the early dark period with a gradual decrease as compared to the SAD, which demonstrated a uniformly lower pattern of consumption. The reduction in intake in the SAD appeared to be specific for NaCl because there was no difference in water or sucrose intake. The deficit could not be attributed to alterations in food intake, nor was there any difference in the amount of water consumed after the saline challenge.

Chronotoxicity of Methotrexate in Mice and Its Relation to Circadian Rhythm of DNA Synthesis and Pharmacokinetics

The mechanisms underlying the circadian rhythm of methotrexate (MTX)-induced toxicity (body weight loss and leukopenia) were investigated from the viewpoints of the sensitivity of living organisms to the drug and the pharmacokinetics of the drug. ICR male mice were housed in a standardized light-dark cycle (lights on at 0700, off at 1900) with food and water ad libitum. The body weight loss after an intraperitoneal injection of MTX (400 mg/kg) was more serious in the late dark period and the early light period and milder in the late light period and the early dark period. The MTX-induced leukopenia was more serious in the late dark period and the light period and milder in the early dark period. Lower toxicity was observed when DNA synthesis, dihydrofolate reductase (DHFR) activity in bone marrow cells and folate level in plasma decreased, and higher toxicity was observed when they increased. There was a significant circadian rhythm in plasma MTX concentration, with a higher level in the light period and a lower level in the dark period. The circadian rhythm of plasma MTX concentration was associated with that of MTX-induced toxicity. The present study suggests that the circadian rhythm of MTX-induced toxicity is caused by that of the sensitivity of living organisms to the drug and the pharmacokinetics of the drug.

Metergoline potentiates natural feeding and antagonizes the anorectic action of medial hypothalamic 5-hydroxytryptamine

Medial hypothalamic injections of 5-hydroxytryptamine (5-HT) or its agonists have been reported to inhibit feeding elicited by norepinephrine (NE), suggesting that these two transmitter systems interact antagonistically in the control of ingestive behavior. The present study was designed to directly test the hypothesis that 5-HT inhibits adrenergic feeding, specifically at the onset of the rat's nocturnal eating cycle. Free-feeding animals were injected with 5-HT (5–20 nmol) immediately before NE (20 nmol) and food intake was measured 1.5 h postinjection. In separate groups of rats, the serotonergic antagonist metergoline (MET) (2.5–20 nmol) was injected into the paraventricular nucleus (PVN) immediately before 5-HT, or before combined injections of 5-HT and NE. The feeding-stimulant action of MET alone, injected IP (0.25–2 mg/kg) or centrally (2.5–40 nmol), was also examined. Results indicated that administration of 5-HT into the PVN suppressed dark onset feeding and dose-dependently blocked NE-stimulated eating. Pretreatment with MET attenuated the inhibitory action of 5-HT on feeding, and reversed the serotonergic blockade of the adrenergic eating response. Further, systemically injected MET significantly increased dark onset feeding, whereas PVN injections failed to alter food intake reliably. These findings provide the first direct evidence that serotonergic and adrenergic systems within the PVN interact in a competitive manner to modulate the natural high rates of feeding displayed by rats during the early dark period. Although MET effectively blocked the anorectic effect of 5-HT, the feeding-stimulant action of this compound alone does not appear to be mediated within the PVN.

Buspirone-induced carbohydrate feeding is not influenced by route of administration and nutritional status

Rats were habituated to ad lib food intake from two isoenergetic diets that differed in carbohydrate and protein content. To examine the route of administration effect, buspirone (0.6, 1.0, and 1.4 mg/kg) was injected into satiated rats either subcutaneously or intraperitoneally. Overall, no route of administration effect was observed; however, when results of the lowest dose were analyzed separately, the subcutaneous route was more effective than the intraperitoneal route. Regardless of route of administration, buspirone increased food intake over the first 2 h of food presentation in a dosedependent manner. Moreover, the increase was entirely attributed to increases in intake from the high carbohydrate diet. In the subsequent experiment, the effect of buspirone (0.6 mg/kg) was examined in both satiated (early light period) and nonsatiated rats (early dark period). Both groups responded to buspirone with an increase in carbohydrate intake. Despite differences in baseline intake, the absolute increase was similar between satiated and nonsatiated rats. These data suggest that both sensitivity and selectivity of buspirone-induced feeding are neither influenced by route of administration nor nutritional status of rats.

Effects of adrenalectomy on macronutrient selection patterns in the rat

The present studies examined the effects of adrenalectomy (ADX) on nutrient selection of rats over the 24-h period, as well as during the first 2 h of the nocturnal feeding cycle. Results indicate that ADX, in rats showing generally similar preferences for carbohydrate and fat, equally suppresses intake of both of these nutrients over the 24-h period. The relative impact of ADX on carbohydrate and fat intake may shift depending upon baseline, with carbohydrate-preferring rats showing a stronger decrease in intake of this diet after ADX and fat-preferring rats exhibiting a greater decline in fat intake after ADX. Acute injections of corticosterone (CORT) and aldosterone (ALDO) are both found to restore carbohydrate as well as fat intake to ADX rats over the 24-h period. However, in the first 2 h of the dark feeding cycle, carbohydrate intake is found to be selectively suppressed after ADX, and CORT injection (0.5 and 2.0 mg/kg, SC) restores carbohydrate intake during this early dark period, while producing a small increase in fat intake only at the higher dose. This is in contrast to ALDO administration at dark onset, which has a stronger stimulatory effect on fat intake in the ADX rat but does not fully restore carbohydrate intake. These findings indicate that CORT and ALDO have differential effects on nutrient intake in ADX rats particularly at the onset of the dark cycle, and it is suggested that these effects are mediated, respectively, by the type I and type II steroid receptor systems in the brain.

Effect of hypothalamic and peripheral fluoxetine injection on natural patterns of macronutrient intake in the rat

The effect of the serotonin (5-HT) reuptake inhibitor, fluoxetine (FLU), on nutrient intake was examined in rats given free access to three pure macronutrient diets (protein, carbohydrate and fat). Fluoxetine was administered either peripherally or directly into the hypothalamic paraventricular nucleus (PVN) at three different times of the rats' nocturnal cycle. Using a range of doses for IP (0.6-10 mg/kg) and PVN injection (3.2-100 nmol), FLU exerted a selective, dose-dependent suppression (-20% to -60%) of carbohydrate intake only during the first hour of the dark. No change in the consumption of protein or fat was observed. This suppressive effect in the early dark period was not observed during the late dark phase, after either IP or PVN administration. In animals with brain cannulae aimed at different hypothalamic nuclei, the nutrient-suppressive effect of FLU was found to be localized to the medial hypothalamic nuclei, namely, the ventromedial, dorsomedial and suprachiasmatic nuclei, in addition to the PVN. These results, along with other published work, support a role for hypothalamic 5-HT systems in the control of nutrient intake in a circadian-related manner and in mediating the central action of the anorectic compound FLU.

Self-selecting albino rats exhibit differential preferences for pure macronutrient diets: Characterization of three subpopulations

Analyses of natural feeding behavior in albino male Sprague-Dawley rats demonstrate that, when allowed to self-select from pure macronutrient diets (protein, carbohydrate and fat), these rats of the same genetic strain can be categorized into 3 subpopulations according to either their 24-h or their 12-h nocturnal patterns of nutrient intake. A majority of the animals (HC for high carbohydrate, 50% of the total population) consumed a diet rich in carbohydrate relative to protein or fat, while a smaller population of rats (HF, 30%) preferred the fat diet, and an even smaller population (HP, 20%) chose a high-protein diet. These 3 subpopulations, after a few weeks of maintenance on the diets, differed in their body weight, with the HF rats having a higher body weight than the HP animals, who tended to weigh more than the lightest HC rats. Whereas all subgroups exhibited a similar bimodal distribution of feeding during the nocturnal cycle, with peaks during the early and late dark periods, they were distinguishable on the basis of their nutrient consumption during specific phases of the dark cycle. This difference was most apparent in the early dark phase, when the 3 subgroups exhibited exaggerated preferences for the specific nutrient that was generally preferred over the 24-h cycle. This is in contrast to the middle dark phase, when diet preferences were attenuated or lost, and the late dark phase, when most rats were similar in showing an increased preference for protein and fat and a decreased preference for carbohydrate. The HF group was further distinguished by an unusually strong burst of feeding during the first 2 h of the dark period and an extra peak of feeding in the middle dark period (7th h), both of which were relatively high in fat content.

Source of Respiratory Carbon Evolved from the Shoots of Rice Seedlings in Darkness.

The time course change of the shoot respiration rate, the percentage of currently assimilated carbon in respiration and the amounts of current and reserved compounds in the shoots of rice plants were examined using the steady-state 13CO2 assimilation technique, in an attempt to evaluate the change of respiratory substrates with time during darkness. The shoot respiration exhibited midnight rise of respiration rate (MRR), and then a sudden decrease around 13 hours after the onset of darkness. The percentage of CO2 derived from 13C-labeled current assimilates in respiration was high during the first 6 hours, and followed by a continuous decrease. Therefore, current assimilates took priority over reserved compounds to support midnight respiration with MRR in the dark period, and as being consumed current photosynthates the respiration using reserved materials increased to compensate the respiration. Since the 13C-labeling and the content of sucrose were high during the early dark period, it was suggested that sucrose was the principal source of respiratory substrates, and thereafter, starch and other compounds gradually became the major source of CO2. The integrated value of the percentage of 13C-labeled CO2 in total respired CO2 during the first 12 hours was 51%, suggesting respiration dependency on current assimilates was almost equivalent to that on reserved materials.

雌ラットの性成熟過程における行動活性リズムの変化

Adult female rats showed increase in ambulatory activity and marked decrease in drinking behavior at early dark period (18:00-24:00) of proestrus stage before ovulation. In this study, we observed the changes in patterns of ambulatory and drinking activities of immature rats associated with estrous cycle during growing period. Immature female rats of the Wistar-Imamichi strain were used, and both behavioral activities were measured continuously, using Gundai type Ambulodrinkometer. From these observations, it was found that the typical patterns of ambulatory and drinking activities which were observed in adult female rats appeared at the sixth (51-60 days old) and the third (39-49 days old) estrous cycle, respectively.

Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN

The feeding responses elicited by injection of norepinephrine (NE), neuropeptide Y (NPY) and galanin (GAL) into the paraventricular nucleus (PVN) were studied at two different times of the dark (active) cycle in male Sprague-Dawley rats maintained ad lib on pure nutrient diets. The feeding response elicited by NE in the PVN, characterized by a potent and selective stimulatory effect on ingestion of the carbohydrate diet, was significantly stronger during the early dark period (+11.7 kcal over vehicle baseline) relative to the late dark period (+7.6 kcal). A similar pattern of effects was observed with NPY in the PVN, which also selectively potentiated carbohydrate ingestion. The effects of GAL were different from those observed with NE and NPY. Whereas the total amount of food consumed after PVN GAL injection was similar in the early and late dark periods, the macronutrient selection patterns exhibited at these two times were different. During the early dark period, PVN GAL had a small stimulatory effect on carbohydrate, in addition to a strong enhancement of fat intake; in the late dark period, in contrast, GAL stimulated intake only of the fat diet. These findings may reflect differential functions of these hypothalamic neurotransmitters in controlling nutrient ingestion at different periods of the circadian cycle.

Circadian Rhythm of Oxytocin in the Cerebrospinal Fluid of Rhesus and Cynomolgus Monkeys: Effects of Castration and Adrenalectomy and Presence of a Caudal-Rostral Gradient

Oxytocin and vasopressin (AVP) were previously reported to have a diurnal rhythm in cerebrospinal fluid (CSF) collected from the cervical cistern of chaired, intact male rhesus monkeys. In the present study, we continuously sampled CSF from temporary indwelling catheters placed in the spinal subarachnoid space of unanesthetized monkeys maintained on tether and swivel systems. CSF was collected from intact and castrate female rhesus monkeys and intact female and castrate, adrenalectomized male cynomolgus monkeys to determine if oxytocin and AVP rhythms are expressed in spinal subarachnoid CSF, if the magnitude of the CSF rhythm displays a rostral-caudal gradient, and if the rhythm is present in adrenalectomized and castrate monkeys, or is specific to the sex or species of macaque. Monkeys, maintained on a 12-hour light/dark cycle with lights on from 06.00 to 18.00 h, had 19-gauge epidural catheters introduced at the L4-L5 intervertebral space and advanced cephalad in the subarachnoid space. The proximal end of the catheter was connected to a peristaltic pump for continuous removal of CSF (0.5 ml/h) and hourly CSF samples were radioimmunoassayed for oxytocin and AVP. For rostral-caudal studies, the distal tip of the catheter was repositioned every few days to collect CSF from 3 levels of the spinal subarachnoid space: C5-6, T5-6, T12-L1. Each animal had a diurnal CSF oxytocin rhythm with peak and trough oxytocin levels during early light and dark periods, respectively. The magnitude of the oxytocin rhythm differed among animals, but was consistent in an animal from day to day.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors.

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significantly proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.

Twenty-four hour variations in subcellular structures of rat pancreatic islet B-, A- and D-cells, and of portal plasma glucose and insulin levels.

Variations in stomach weights, plasma glucose and insulin levels in the portal vein, and in subcellular structures of rat pancreatic islet B-, A- and D-cells were examined over 24 h. The variation in stomach weights paralleled plasma glucose levels, indicating that the levels may be influenced by intestinal glucose absorption. Plasma insulin levels increased from the late dark to the early light periods, whereas they decreased from the late light to the early dark periods. The variation in plasma insulin levels was in the opposite sense to that in the relative numbers of B-cell granules. The decrease in the relative numbers of A-cell granules occurred between the late dark and early light periods. The relative numbers of D-cell granules decreased before and after the decreases in B- and A-cell granules. The variation in D-cell granules appears to correspond to the inhibitory effect of somatostatin. The relative amounts of rough endoplasmic reticulum (rER) in each cell varied in a reciprocal manner to those of B-cell granules. Moreover, the variation in plasma insulin levels coincided with variations in rER of hepatocytes and pancreatic acinar cells. The changes in rER of each cell may correlate with the trophic effect of insulin.

The pineal gland of the Mongolian gerbil: nocturnal increase of electrical activity.

Extracellular single unit recordings were made during day- and nighttime from spontaneously active pineal gland cells in male Mongolian gerbils. Units exhibited an irregular firing pattern. Discharge frequencies ranged from 0.1 to 8.5 Hz and showed distinct time-dependent differences. During daytime, mean firing frequencies were in the range of 1 Hz and increased to 2 Hz in the early dark period, reaching a peak (ca. 5 Hz) in the middle of the night, followed by a decrease to 2 Hz in late night. The results of this first report on pineal electrical activity in Mongolian gerbils are discussed in comparison to those obtained from rat, guinea pig and golden hamster.

Impact of acute corticosterone administration on feeding and macronutrient self-selection patterns.

Food intake, body weight, and meal patterns in rats are known to be specifically influenced by circulating corticosterone (CORT). The present study examined, in Sprague-Dawley rats, the role of CORT in the regulation of caloric intake and choice of macronutrients during specific periods of the light-dark cycle. Adrenalectomy (ADX) significantly attenuated the ingestion of all three macronutrients, namely, protein, carbohydrate, and fat, in different test periods of the light-dark cycle. This deficit was significantly stronger just before or during the early dark hours, compared with the light period, and it was immediately CORT reversible. During this same early dark period, the stimulatory effect of CORT (0.5 or 2.0 mg/kg sc) on total food intake in ADX animals was also significantly stronger than during the light period. At this time, the CORT-injected ADX animals consumed an even greater amount of total food intake than did the vehicle-injected sham-operated animals and showed a significant preference for carbohydrate, as opposed to no change or a decline in preference for protein or fat. These findings, in light of other evidence, suggest that CORT ensures and stabilizes the ingestion of all three macronutrients but, in particular, stimulates carbohydrate ingestion during the important feeding period at the dark onset when CORT levels normally peak. It is proposed that CORT may provide a critical signal, in conjunction with hypothalamic neurotransmitter systems, in the behavioral and metabolic regulation of body energy balance.

On the daily variation in the beta-receptor-adenylate cyclase-cAMP-phosphodiesterase system in rat forebrain.

In rat forebrain tissue of single rats beta-adrenoceptor density (Bmax) and affinity (Kd) were determined by saturation isotherms in receptor binding studies with the antagonist ligand (3H)-dihydroalprenolol at 8 different times of day in May. Rats were on a controlled 12L:12D photoperiod. In addition, the cAMP content, the formation of cAMP from ATP by the adenylate cyclase and the hydrolysis of the second messenger by the phosphodiesterase were determined at the same time points. No significant (ANOVA) daily variations were found in the total number of 3H-DHA binding sites (Bmax) nor in the affinity (Kd). In contrast, basal cAMP content as well as basal formation and hydrolysis of cAMP displayed significant rhythms. The peak value in cAMP was at the beginning of light. At that time the daily trough value in cAMP formation was found. Hydrolysis of cAMP by the phosphodiesterase displayed a 12-hr rhythm with trough values occurring at the early light and early dark period. The results demonstrate pronounced rhythmic changes in basal formation, content and hydrolysis of cAMP which are, however, not paralleled by changes in receptor number and/or affinity in the same tissue.

Autophagic vacuoles in heart muscle and liver. A comparative morphometric study including circadian variations in meal-fed rats

Large test areas of 36 000 to 38 000 μm 2 of scctioned cytoplasm/animal and organ were morphometrically evaluated for autophagic vacuoles (AVs) in heart muscle and in liver parenchymal cells. Rats were kept in a nutritional steady state by feeding a timely, i.e. shortly before the onset of darkness, single daily meal (10 g of a standard diet). Circadian variations in volume fraction as well as in numerical density of AVs were found. The values reached a maximum in the late light period, whereas minimal values were obtained in the early dark period. The variations were synchronous in heart muscle and in liver. The average cytoplasmic volume fraction of AVs in heart muscle was 0.79 × 10 −4, that is less than half the value found in liver 1.74 × 10 −4. The differences were less pronounced in the segregated fraction of mitochondria which amounted to 1.68 × 10 −4 in heart muscle and 2.85 × 10 −4 in the liver. These data suggest that in heart muscle cytoplasmic components are degraded by cellular autophagy to a similar, perhaps some lower rate than in liver. The absence of myofibrils in the AVs does not exclude a possible lysosomal degradation of myofibrillar proteins.